This project seeks to clarify the molecular and cellular mechanisms by which Presenilins (PS1 and PS2) and associated molecules mediate protein trafficking, gamma-secretase activity and AB production. Recent studies have revealed that high molecular weight protein complexes containing PS play a critical role in AB production. However, the molecular composition of PS1 complexes has not been elucidated. In this regard, a type I membrane protein, termed nicastrin, was recently identified as a stoichiometric component of the PS1 complex. Experimental mutants of nicastrin effect AB production and Notch 1 cleavage, suggesting an accessory role for this molecule in regulating gamma secretase activity. Under Aim 1, we propose to characterize the cellular and subcellular distributions of nicastrin, the interaction of nicastrin and PS1 using deletion mutagenesis strategies, and assessment of the role of PS1-nicastrin interactions on gamma-secretase activity. Finally, we will investigate the co-distribution of APP, APP B-CTF, PS1 and nicastrin by biochemical and immunocytochemical methods. In addition to its role in mediating intramembranous gamma-secretase cleavage of APP and Notch, PS1 is also known to regulate the trafficking of several membrane proteins, including APP and the neurotrophin receptor, TrkB. Our Preliminary Results show that experimental PS1 mutants that effect APP processing also alter the subcellular distributions of APP, APP CTFs, APLP2 and TrkC. Studies under Aim 2 will extend these investigations to examine the role of PS1 on the trafficking of APP, APP homologues, neurotrophin receptors, low-density lipoprotein receptor-related protein (LRP), transferrin receptor, and AMPA receptors. Under Aim 3, we outline biochemical strategies to identify and characterize the molecular components of PS1 complexes and our approaches to characterize gamma-secretase activity in vitro. These latter studies will also facilitate the identification of novel substrates of gamma-secretase. Collectively, the studies proposed will provide new insights into: the functional relationship between nicastrin and PS1 in modulation of gamma-secretase activity; the identity of components contained within PS1-resident high molecular weight complexes; and the function of PS1 in regulating intracellular trafficking and metabolism of membrane proteins.